PROJECT SUMMARY Antimicrobial resistance (AMR) is an increasingly prevalent and serious problem worldwide. Most often, AMR arises from horizontal gene transfer (HGT), involving mobile genetic elements (MGE) such as plasmids and phages. The human gut is a hotspot for both the evolution and spread of AMR; commensals serve as a source of AMR for pathogens via HGT. Slowing the evolution and spread of AMR is both possible and necessary. Knowledge about the evolutionary history of AMR development and dissemination in vivo is essential to facilitate effective stewardship. Yet, this knowledge remains limited. Important aspects of AMR evolution become evident only in complex environments and in the setting of diverse communities. We will study responses to antibiotic exposure in the human gut microbiota in vivo, as well as in complex stool-derived subject-specific communities in vitro, at high temporal resolution and using innovative approaches. We will link AMR and other MGE- associated genes to their host core genomes using high throughput chromosome conformation capture (Hi-C) and monitor these genes and elements in bacterial hosts before, during and after antibiotic exposure. The short term-objectives of the proposed work are to characterize and assess the contributions of de novo mutations and HGT to the spread and development of AMR in the human gut microbiota during antibiotic exposure. The long- term objectives are to improve antibiotic stewardship by identifying critical events or transitions in the evolution and dissemination of AMR in vivo, and the factors and conditions that make those events less likely. Aim 1. Determine the distribution of antimicrobial resistance genes in the gut microbiota of healthy humans. We will use Hi-C and metagenomic sequencing to resolve strain-level microbial genomes from stool samples of 60 healthy adults collected over an 8-week antibiotic-free interval, prior to a ciprofloxacin exposure. We will identify potential AMR genes and determine their distribution within core and accessory genomes, and in association with mobile genetic elements. Aim 2. Characterize the effects of ciprofloxacin on the abundance and mobilization of AMR genes in the human gut microbiota in vivo. We will use Hi-C and metagenomic sequencing to assay stool samples collected from the same 60 subjects during and after the ciprofloxacin exposure, and to characterize the composition and dynamics of selective sweeps that affect the emergence of AMR. Aim 3. Characterize the effects of ciprofloxacin on the mobilization of AMR genes in synthetic, human gut-derived microbial communities in vitro. We will propagate pre-exposure fecal communities ex vivo from all 60 subjects, as well as generate complex, synthetic communities from pre-exposure samples of 5 subjects, and then passage both bulk and synthetic communities anaerobically under multiple ciprofloxacin regimes. We will identify factors and conditions that affect emergence of AMR through HGT and de novo mutations in vitro.